Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Cytosolic Phospholipase A2M-NM-1 and Eicosanoids Regulate Expression of Genes in Macrophages Involved in Host Defense and Inflammation

ABSTRACT: The role of Group IVA cytosolic phospholipase A2 (cPLA2M-NM-1) activation in regulating macrophage transcriptional responses to Candida albicans infection was investigated. cPLA2M-NM-1 releases arachidonic acid for the production of eicosanoids. In mouse resident peritoneal macrophages, prostacyclin, prostaglandin E2 and leukotriene C4 were produced within minutes of C. albicans addition before cyclooxygenase 2 expression. The production of TNFa was lower in C. albicans-stimulated cPLA2a+/+ than cPLA2a-/- macrophages due to an autocrine effect of prostaglandins that increased cAMP to a greater extent in cPLA2a+/+ than cPLA2a-/- macrophages. For global insight, differential gene expression in C. albicans-stimulated cPLA2a+/+ and cPLA2a-/- macrophages (3 h) was compared by microarray. cPLA2M-NM-1+/+ macrophages expressed 86 genes at lower levels and 181 genes at higher levels than cPLA2M-NM-1-/- macrophages (M-bM-^IM-%2-fold, p<0.05 ). Several pro-inflammatory genes were expressed at lower levels (Tnfa, Cx3cl1, Cd40, Ifng, several IFNg-inducible GTPases, Ccl5, Csf1, Edn1, CxCr7, Irf1, Irf4, Akna). Genes that dampen inflammation (Socs3, Il10, Crem, Stat3, Thbd, Thbs1, Abca1) and genes involved in host defense (Gja1, Csf3, Trem1, Hdc) were expressed at higher levels in cPLA2a+/+ macrophages. Representative genes expressed lower in cPLA2a+/+ macrophages (Tnfa and Csf1) were increased by treatment with a prostacyclin receptor antagonist and protein kinase A inhibitor, whereas genes expressed at higher levels (Crem, Nr4a2, Il10, Csf3) were suppressed. The results suggest that C. albicans stimulates an autocrine loop in macrophages involving cPLA2a, cyclooxygenase 1-derived prostaglandins and increased cAMP that globally effects expression of genes involved in host defense and inflammation. Resident peritoneal macrophages from Group IVA cytosolic phospholipase A2 (cPLA2a) +/+ and -/- mice were stimulated with Candida albicans (m.o.i. 2) for 3 hours and RNA isolated to evaluate the role of cPLA2a-derived lipid mediators on gene expression. Three independent experiments were analyzed.

ORGANISM(S): Mus musculus

SUBMITTER: Christina Leslie

PROVIDER: E-GEOD-46533 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Lipid A (a hexaacylated 1,4 bis-phosphate) is a potent immune stimulant for TLR4/MD-2. Upon lipid A ligation, the TLR4/MD-2 complex dimerizes and initiates signal transduction. Historically, studies also suggested the existence of TLR4/MD-2-independent LPS signaling. Here we define the role of TLR4 and MD-2 in LPS signaling by using genome wide expression profiling in TLR4- and MD-2-deficient macrophages after stimulations with peptidoglycan-free LPS and synthetic E.coli lipid A. Of the 1,396 genes found significantly induced or repressed by any one of the treatments in the wildtype macrophages, none was present in the TLR4- or MD-2-deficient macrophages, confirming that the TLR4/MD-2 complex is the only receptor for endotoxin, and are both absolutely required for responses to LPS. Using a molecular genetics approach, we investigated the mechanism of TLR4/MD-2 activation by combining the known crystal structure of TLR4/MD-2 with computer modeling. We used lipid IVa, a defined lipid A mimetic to model the activation of mouse TLR4/MD2. The two phosphates on lipid A were predicted to interact extensively with the two positively charged patches mouse TLR4 according to our dimeric murine TLR4/MD-2/lipid IVa model. These two patches are composed of K263, R337, and K360 (Positive Patch 1), and K367 and R434 (Positive Patch 2). When either Positive Patch was abolished by mutagenesis into Ala, the responses to LPS and lipid A were almost abrogated. Thus, ionic interactions between the two phosphates on lipid A and the two positively charged patches on murine TLR4 appear to be essential for LPS receptor activation. The gene expression profile of macrophages from C57BL/6 and MD-2-deficient mice following either 10 ng LPS /mL, 100 ng lipid A/mL or 10 nM Pam2 stimulation for 2 hours were compared to PBS-stimulated control cells . In vitro differentiated macrophages from two individual WT and MD-2-deficient mice were cultured and stimulated with agonists separately, comparing the gene expression to PBS-stimulated control cells from the same mouse. Comparisons of PBS-stimulated WT cells to PBS-stimulated MD-2-deficient cells were performed to directly compare basal gene expression in the two genotypes.

Project description:Lipid A (a hexaacylated 1,4 bis-phosphate) is a potent immune stimulant for TLR4/MD-2. Upon lipid A ligation, the TLR4/MD-2 complex dimerizes and initiates signal transduction. Historically, studies also suggested the existence of TLR4/MD-2-independent LPS signaling. Here we define the role of TLR4 and MD-2 in LPS signaling by using genome wide expression profiling in TLR4- and MD-2-deficient macrophages after stimulations with peptidoglycan-free LPS and synthetic E.coli lipid A. Of the 1,396 genes found significantly induced or repressed by any one of the treatments in the wildtype macrophages, none was present in the TLR4- or MD-2-deficient macrophages, confirming that the TLR4/MD-2 complex is the only receptor for endotoxin, and are both absolutely required for responses to LPS. Using a molecular genetics approach, we investigated the mechanism of TLR4/MD-2 activation by combining the known crystal structure of TLR4/MD-2 with computer modeling. We used lipid IVa, a defined lipid A mimetic to model the activation of mouse TLR4/MD2. The two phosphates on lipid A were predicted to interact extensively with the two positively charged patches mouse TLR4 according to our dimeric murine TLR4/MD-2/lipid IVa model. These two patches are composed of K263, R337, and K360 (Positive Patch 1), and K367 and R434 (Positive Patch 2). When either Positive Patch was abolished by mutagenesis into Ala, the responses to LPS and lipid A were almost abrogated. Thus, ionic interactions between the two phosphates on lipid A and the two positively charged patches on murine TLR4 appear to be essential for LPS receptor activation. Bone marrow-derived macrophages were pooled from four individual WT or TLR4-deficient mice and stimulated with either 10 ng LPS /mL, 100 ng lipid A/mL or 10 nM Pam2 for 2 hours and compared to PBS-stimulated control cells. We also compared PBS-stimulated WT cells directly to PBS-stimulated TLR4-deficient cells to compare the basal expression of genes in the two genotypes. This experiment was repeated once in its entirety.

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Cytosolic Phospholipase A2M-NM-1 and Eicosanoids Regulate Expression of Genes in Macrophages Involved in Host Defense and Inflammation

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